Skin pigmentation levels and regulation are determined and controlled by a complex system of interrelated genes, proteins and a milieu of intracellular and extracellular compounds, some of which have yet to be elucidated. Skin models capable of mimicking aspects of cellular processes integral to the skin pigmentation pathway(s) are therefore desired in order, for example, to identify skin-active agents effective in modulating skin pigmentation.
Modeling techniques to study skin physiology and skin responses to agents have historically included a variety of specific techniques, from the culturing of a single cell type or a small number of co-mingled cell types, to fabricating human tissue equivalents, to developing animal models. However these relatively simple models often lack much of the intra and intercellular complexities of human skin. For example, cell cultures of single cell types are easily utilized but overly simplistic and have severe limitations for generalizing results to human skin. First, such cultures contain cells that are altered simply by being cultured and, in specific cases, have been altered by genetic manipulation to promote easy cell passaging and maintenance. Second, such cultures cannot account for intrinsic intricate matrices of cells constantly interacting as a unit. Multi-celled cultures are limited due to difficulties in creating the integrated mechanical structure of native tissue and in ensuring that the cells comprise the extracellular components necessary to maintain cellular genetic expression levels at a normal physiological level. Models that include stem cells treated to mimic human skin are suitable for their intended purposes, but fall victim to similar concerns and limitations.
More complex skin models include animal models and skin-equivalent models. While having additional complexity, animal models suffer from limitations including the genetic variation with respect to human skin; in the analysis of obtained results there is always a concern that human tissues react differently from animal tissues and the ability to generalize results is compromised. For example, animal models of skin may differ substantially from human skin, in natural pigment levels or in the ability to up-regulate specific genes. Additionally, even slight variations in receptor levels can lead to responses that are less, more, or seemingly idiosyncratic in relation to what might be seen in human skin responses depending on whether the tested agent binds with different kinetics or not at all, or in such a way as to activate or inactivate various alternative internal pathways or genes. In addition, extrinsic factors can affect test results with animals, and stressors unrelated to the test agent could also affect results.
Skin-equivalent models are limited by lack of cellular interconnectivity, permeability concerns, and anatomical simplicity. More recent attempts at skin-equivalent models may be described as organotypic human tissue equivalents and include in vitro reconstructions of human cells such as keratinocytes cultured on an inert polycarbonate filter. These models by their very nature are limited in that they can have reduced barrier function that can lead to aberrant sensitivities to tested agents. The models also are less complex than human skin, having perhaps one or two cells types (such as keratinocytes and fibroblasts or keratinocytes and melanocytes) but lacking additional cells such as endothelial cells or even the full keratinocyte, fibroblast, and melanocyte combination. In addition the organotypic skin equivalent models are also missing normal skin structures such as glands that can affect skin response.
The most complex skin model involves the ex vivo culture of human skin tissue samples. Previous attempts to utilize ex-vivo human skin as an assay model had limitations such as brief life-spans with low vitality or viability. Previous attempts at such models included small biopsies of skin floating directly in media, which is not analogous to the normal environment of the skin and resulted in the tissue having a limited lifespan. It is known in the art that transient cultures can be deficient, as inventors and researchers have indicated attempts at ex-vivo pig skin grafts are limited to seven days (Vielhaber et al., Ex vivo Human Skin Model, US2009/0298113).
Even more recent ex-vivo models have involved attempting to culture skin explants on metal grids (Mitts et al., Elastin Protective Polyphenolics and Methods of Using the Same, US2009/0110709) and skin grafting to the chorioallantoic membrane (CAM) of a fertilized ovarian egg (Goldstein et al., Chimeric Avian-Based Screening System Containing Mammalian Grafts, US2009/0064349). However such models are still limited by transiency of the construct, delicacy, and even xenogeneic concerns. One attempt at improving the longevity of ex-vivo skin is described in EP 2 019 316 B1.
Despite these advancements, a need continues to exist for a sensitive and predictive ex-vivo human skin tissue screening method that is robust enough to manage inherent donor-to-donor tissue variability, is stable over a sufficient time period to identify both weak and strong tone agents, and is predictive based upon an analysis of the gene transcriptomics of the donor tissue. The latter is particularly useful in a low through-put screening method for tone agents, as measuring gene transcriptomics is relatively quick and fold changes are reliably ascertainable over shorter time periods more than other end points, such as measuring the amount of melanin produced or inhibited in the ex-vivo tissue sample.
As discussed more fully hereafter, the challenges and uncertainties associated with developing a practical screening method for tone agents are numerous. Non-limiting examples include: the effect of donor to donor skin type variability, the effect of donor to donor skin status variability (e.g., the state of inflammation and viability) post surgical extraction, whether culturing conditions could establish viability of the tissue samples for sufficient periods of time to assess both slow and fast acting tone agents, whether ex-vivo skin tissue can be properly regulated at the gene transcriptomic level over the needed time periods, whether analysis of gene transcriptomics of ex-vivo skin tissue could be predictive of in vivo results, whether appropriate positive controls could be identified for use in a high enough percentage of the tissue samples to develop a satisfactory screening method, and whether these variables could be controlled to the point that the effects of a tone agent on an ex-vivo skin tissue sample could be isolated and interpreted so as to be repeatable to a level of statistical significance.